Background We recently completed the GWAS studies in AMD and Fuchs corenal dystrophy (FCD). We are continuing our efforts to investigate retinal aging and its impact on common aging-associated diseases. We are employing comprehensive and collaborative approaches and using human genetics along with mouse and zebrafish animal models. Results 1. Aging 1.1. Variation in the apolipoprotein E gene (APOE), a lipid transport protein involved in low-density cholesterol modulation has been reported to be associated with longevity in humans. As part of a multi-center international study, we assessed the allelic distribution of APOE isoforms &#949;2, &#949;3, and &#949;4 among 10,623 control subjects of European ancestry from 15 case-control and cohort studies of age-related macular degeneration (AMD). In an analysis stratified by study center, gender, and smoking status, we found a decreasing frequency of the APOE &#949;4 isoform with increasing age, with a concomitant increase in the &#949;3 isoform. The association with age was strongest in &#949;4 homozygotes;the frequency of &#949;4 homozygosity decreased from 2.7% for participants aged 60 years or less to 0.8% for those over age 85 years, while the proportion of participants with the &#949;3/&#949;4 genotype decreased from 26.8% to 17.5% across the same age range. However, gender had no significant effect on the isoform frequencies. Thus, we provide strong support for an association of the APOE gene with human longevity. 1.2. Advanced age contributes to the clinical manifestation of many retinopathies. Rod photoreceptors are especially vulnerable to genetic defects and changes in microenvironment, and are among the first neurons that are affected in normal aging and in many retinal degenerative diseases. We took advantage of mice expressing eGFP specifically in rod photoreceptors in the retina (Nrlp-eGFP mice) to identify candidate genes and pathways underlying rod vulnerability to aging. We show that changes in metabolic and signaling pathways, including consensus pathways implicated in aging, are affected in young adult mice. These studies support the hypothesis that relevant changes in gene expression and cell metabolism occur well before pathological manifestations of aging, such as reduction in outer nuclear layer thickness and/or alterations in electroretinogram recording, become apparent. Some of the age-related pathways we identify, such as oxidative phosphorylation, regulation of inflammatory response, and transcriptional regulation, are common to neuronal aging. Moreover, we have identified cellular processes specific to retinal aging, such as lipid metabolism and angiogenesis. Hence, changes in gene expression might represent an adaptive response of rod photoreceptors to microenvironment (such as exposure to light and oxidative stress) during the aging process. 2. Age-related macular degeneration (AMD) 2.1. Previous studies report inconsistent associations between AMD and APOE, with potential interaction between APOE and sex, and smoking status. As part of the multicenter international study described above, we performed a pooled analysis (n = 21,160) demonstrating associations between late AMD and APO&#949;4 and APO&#949;2, following adjustment for age-group and sex within each study and smoking status. We found no evidence of interaction between APOE and sex or smoking. Ever smokers had significant increased risk relative to never smokers for both neovascular and atrophic AMD but not early AMD, implicating smoking as a major contributing factor to disease progression from early signs to the visually disabling late forms. Extended haplotype analysis incorporating rs405509 did not identify additional risks beyond &#949;2 and &#949;4 haplotypes. 2.2. Despite extensive association studies, precise genetic variants that cause AMD have not been defined. We are using whole exome capture approach for identifying disease-causing variants in AMD families. We successfully obtained >90% coverage in all samples with an average depth of 130X. This was followed by filtering of variants to exclude common variants identified in the dbSNP or 1000 genome project as well as to select for variants identical-by-descent in the affected siblings. We are currently validating the sequencing variants for segregation in additional family members using Sanger sequencing. We are also searching this comprehensive landscape of coding variants for additional information, which might possibly explain phenotypic heterogeneity and genetic epistasis seen in diseases. 2.3 We are also involved in a major medical re-sequencing project at Washington University with colleagues at Michigan and UPenn to define the causal variants in the genetic loci associated with AMD. Specifically, we are sequencing genomic regions (from 100 kb to about 1 Mb) flanking the AMD-associated SNPs at 7-10 different loci. Dr. Abecasis will analyze the data and we will focus on validation and biology to understand AMD etiology. 2.4 Further to our GWAS, we performed a prospective case-control study with a Spanish cohort of 353 cases with advanced AMD and 282 controls in collaboration with colleagues at Michigan and in Spain. In agreement with our previous data, ARMS2 and CFH genes appear to be the principal risk loci contributing independently to AMD in the Spanish population. Both loci influence risk independently, giving support to different pathways implicated in AMD pathogenesis. Two new associations could influence the development of advanced AMD: a variant located at the 3'UTR of the FGF2 gene (rs6820411), highly associated with atrophic AMD: and the functional SNP rs3112831 at ABCA4, showing marginal association with the disease. These latter findings require validation with larger cohorts. 2.5 We have established and contributed to a GWAS meta-analysis consortium for AMD genetic studies. Almost 20 different groups from all over the world are participating in this international collaborative effort. Initial analysis has now identified 19 distinct AMD genetic loci. The results are being prepared for a major publication. 2.6 From the gene list derived from the recent AMD-GWAS meta-analysis, we have selected a subgroup of 10 genes that we are being tested by gain or loss of function studies in zebrafish. The goal is to determine if any of AMD loci plays a direct role in eye/retina development or function. 3. Diabetic Retinopathy Diabetic retinopathy (DR) is the leading cause of blindness in working-age Americans. The Candidate-gene Association Resource (CARe) is a multicentric international collaboration for association analyses of genotypes and cardiovascular disease phenotypes. It comprises >40,000 participants from nine cohorts who have been genotyped for 49,320 single nucleotide polymorphisms (SNPs) from approximately 2,000 candidate genes postulated or known to increase risk of cardiovascular, metabolic and inflammatory diseases. We investigated whether variants in cardiovascular candidate genes, some of which have been previously associated with T2D, DR, and DN, are associated with DR in the CARe. Among 39 genes previously associated with DR, DN or T2D, three SNPs in P selectin (SELP) were associated with DR. The strongest association was to rs6128. These associations remained significant after adjusting for DR risk factors. Further studies are in progress.